The present invention provides new psoralens having enhanced ability to inactivate pathogens in the presence of ultraviolet light. The present invention also provides methods of using new psoralens to inactivate pathogens in health related products to be used in vivo and in vitro, and in particular, blood products.
Psoralens are tricyclic compounds formed by the linear fusion of a furan ring with a coumarin. Psoralens can intercalate between the base pairs of double-stranded nucleic acids (or base paired regions of single-stranded nucleic acids), forming covalent adducts to pyrimidine bases upon absorption of long wave ultraviolet light (UVA). G. D. Cimino et al., Ann. Rev. Biochem. 54:1151 (1985); Hearst et al., Quart. Rev. Biophys. 17:1 (1984). If there is a second pyrimidine adjacent to a psoralen-pyrimidine monoadduct and on the opposite strand, absorption of a second photon can lead to formation of a diadduct which functions as an interstrand crosslink [S. T. Isaacs et al., Biochemistry 16:1058 (1977); S. T. Isaacs et al., Trends in Photobiology (Plenum) pp. 279-294 (1982); J. Tessman et al., Biochem. 24:1669 (1985); Hearst et al., U.S. Pat. Nos. 4,124,598, 4,169,204, and 4,196,281, hereby incorporated by reference].
The photoreaction of psoralens with nucleic acid has been useful in the study of nucleic acid folding, the attachment of diagnostic probes tolnucleic acids, the attachment of nucleic acids to surfaces and materials, the blocking of polymerase reactions and the inactivation of organisms and cells that require nucleic acid replication to proliferate, e.g., bacteria, viruses, leukocytes and overproliferating cells, such as those resulting in psoriasis, restenosis, or cancer. The inactivation of a virus can also be applied to preparation of vaccines. The level of reaction with cellular nucleic acid can be modulated to stop proliferation of the cell yet maintain cell functions such as protein synthesis. This can be applied to the treatment of T-cell lymphocytes as a means of preventing graft vs. host disease in, for example, bone marrow transplants.
The use of psoralens for pathogen inactivation in blood products is of particular interest as the safety of the blood supply is an issue of universal concern. While transfusion associated viral infections have been considerably reduced by testing, transmission of human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV) continue to occur in {fraction (1/450,000)} to 660,000 units, {fraction (1/200,000)}) units and {fraction (1/3000)} units respectively [R. Dodd, Blood Supply: Risks, Perceptions, and Prospects for the Future, S. J. Nance, ed., p.1 (1994); E. Lackritz et al., New Eng. J. Med. 333: 1721 (1995)]. Testing is not an option for some viruses. Cytomegalovirus is commonly found within the blood supply yet is of clinical importance only to immune compromised patients for which infection can be fatal [R. Bowden, Blood Safety: Current Challenges, S. J. Nance, ed., p.201 (1992)]. Universal screening for CMV would lead to a serious reduction in eligible donors and thus a reduction in the national blood supply. Special donor pools must be used for these patients at present. It is also recognized that other unknown viruses or new strains of known viruses may find their way into the blood supply and will not be identified until morbidity or mortality is noted, nor will they be able to be screened out until tests become available. The identification of hepatitis G in blood units is the most recent example of such an occurrence [H. Alter, Transfusion 37: 569 (1997)].
Bacterial contamination, especially of platelet concentrates (PC) has been increasingly recognized as a problem as well. It is estimated that {fraction (1/1,000)} to 2,000 PC units show levels of contamination that results in a septic response [J. Morrow et al., JAMA 266: 555 (1991); M. Blajchman, Blood Safety: Current Challenges, S. J. Nance, ed., p.213 (1992); E. Chiu et al., Transfusion 34: 950 (1994)]. There are at present no screening tests available for blood units for any of the ten or so bacteria that have been associated with fatal transfusion associated sepsis in the United States. While there are potential methods for storage of platelets at lower temperatures to alleviate this problem [U.S. Pat. Nos. 5,827,640 and 5,827,741], inactivation of the bacteria by psoralen would have far less impact on the routine storage of platelets.
Psoralens are ideal candidates for photosensitized, decontamination of platelet concentrates [H. Alter et al., Lancet ii:1446 (1988); L. Lin et al., Blood 74: 517 (1989); C. Hanson, Blood Cells 18: 7 (1992)]. For example, 8-methoxypsoralen (8-MOP) is quite effective at deactivation of a number of bacteria found in platelet concentrates. However, it is not sufficiently active to inactive pathogens with small genomes (i.e., viruses) without using concentrations and irradiation times which damage platelets. The highly active psoralen, 4xe2x80x2-aminomethyl-4,5xe2x80x2,8-trimethylpsoralen (AMT), exhibits excellent photochemical inactivation properties but is highly mutagenic in the absence of light in some bacterial assays [S. Wagner et al., Photochem. Photobio. Meeting Abstract, 55: 113S (1992)]. Other 4xe2x80x2- and 5xe2x80x2-aminomethyl substituted psoralens have been developed which show excellent photochemical inactivation properties with considerable reduction in mutagenicity.
Several patents are directed toward psoralen inactivation of pathogens in blood products [G. Wiesehahn et al., U.S. Pat. Nos. 4,727,027 and 4,748,120, L. Lin et al., U.S. Pat. Nos. 5,288,605, 5,482,828, and 5,709,991, and S. Wollowitz et al., U.S. Pat. No. 5,593,823, hereby incorporated by reference]. P. Morel et al., Blood Cells 18:27 (1992) show that 300 xcexcg/mL of 8-MOP together with ten hours of irradiation with ultraviolet light can effectively inactivate viruses in human serum. Similar studies using 8-MOP and AMT have been reported by other investigators [Dodd R Y, et al., Transfusion 31:483-490 (1991); Margolis-Nunno, H., et al., Thromb Haemostas 65: 1162 (Abstract)(1991)]. Indeed, the photoinactivation of a broad spectrum of microorganisms has been established, including HBV, HCV, and IV. [Hanson C. V., Blood Cells: 18: 7-24 (1992); Alter, H. J., et al., The Lancet ii:1446 (1988); Margolis-Nunno H. et al., Thromb Haemostas 65: 1162 (Abstract) (1991); Lin et al. Transfusion 37: 423 (1997)]. There are clearly a broad class of psoralen compounds effective in the inactivation of pathogens in general and particularly in blood products.
The most highly active psoralen compounds useful for inactivation have amino derivatives on the 4xe2x80x2 and 5xe2x80x2 positions [Wollowitz et al., U.S. Pat. Nos. 5,578,736 and 5,654,443, Kaufman U.S. Pat. No. 4,294,822]. 5-alkoxy and 8-alkoxypsoralens with amino substituents at the 8 or 5 position, respectively, as well as 8-aminomethyl psoralen and 8-aminomethyl-4-methylpsoralen are known [J. Hansen et al., J. Med. Chem. 28: 1001-1010 (1985); Kaufman U.S. Pat. Nos. 4,269,851 and 4,328,239]. The limited data provided for these latter compounds suggested that even the amino substituted alkoxypsoralens have relatively poor photoactivity and that amino substitution at the furan ring is important for high photoactivity. Also, these compounds are formed by methods which offer little flexibility in modifying the ring functionality.
The present invention provides new aminopsoralens with unpredicted photoreactivity with nucleic acids that can be used for nucleic acid probe preparations, preparation of conjugates, inhibition of cell proliferation, inactivation of virus for vaccine preparation, and in particular, for the inactivation of pathogens in blood products. The present invention also provides new routes to the synthesis of aminopsoralens and intermediates that may be useful for providing psoralens conjugated to a variety of other functional groups.
With respect to new compounds, some of the new psoralens are primaryamino-pyrone-linked psoralens comprising a primaryamino group (i.e. xe2x80x94NH2 group) linked to the pyrone ring of the psoralen (3- and 4-carbon atoms) via an alkyl chain optionally containing oxygen and nitrogen atoms, and wherein the psoralen ring may have one or more alkyl groups at other positions. The present invention further contemplates psoralen compounds with a primaryamino substituent on the pyrone ring, comprising: a) a substituent A on the pyrone ring, selected from the group consisting of: xe2x80x94(CH2)uxe2x80x94NH2, xe2x80x94(CH2)wxe2x80x94Jxe2x80x94(CH2)zxe2x80x94NH2, xe2x80x94(CH2)wxe2x80x94Jxe2x80x94(CH2)xxe2x80x94Kxe2x80x94(CH2)zxe2x80x94NH2, and xe2x80x94(CH2)wxe2x80x94Jxe2x80x94(CH2)xxe2x80x94Kxe2x80x94(CH2)yxe2x80x94Lxe2x80x94(CH2)zxe2x80x94NH2; wherein J, K, and L are independently selected from the group consisting of O and NH, in which u is a whole number from 1 to 10, w is a whole number from 1 to 5, x is a whole i: number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and b) substituents B, R3, R4, R5, and R6 on the pyrone ring (the 3- or 4-carbon atom which does not have the primaryamino substituent), 5-, 4xe2x80x2-, 5xe2x80x2- and 8-carbon atoms respectively, independently selected from the group consisting of xe2x80x94H and xe2x80x94(CH2)vCH3, where v is a whole number from 0 to 5; or a salt thereof. Where an element is xe2x80x9cindependently selectedxe2x80x9d from a group, it means that the element need not be the same as other elements chosen from the same group. The structure of these compounds is as follows. 
The present invention further contemplates compounds of the above structure with a primaryamino substituent on the pyrone ring, wherein A is at the 3-carbon atom and B is at the 4-carbon atom, preferably wherein A is selected from the group consisting of xe2x80x94CH2xe2x80x94NH2 and xe2x80x94CH2xe2x80x94Oxe2x80x94(CH2)2xe2x80x94NH2. More specifically, the invention contemplates compounds wherein A is xe2x80x94CH2xe2x80x94NH2, and wherein B, R3, R5, and R6 are xe2x80x94H, and wherein R4 is xe2x80x94CH3; wherein A is xe2x80x94CH2xe2x80x94NH2, and wherein R3 and R5 are xe2x80x94H, and wherein B, R4, and R6 are xe2x80x94CH3; wherein A is xe2x80x94CH2xe2x80x94NH2, and wherein R3 and R4 are xe2x80x94H, and wherein B, R5, and R6 are xe2x80x94CH3; wherein A is xe2x80x94CH2xe2x80x94NH2, and wherein R3 is xe2x80x94H, and wherein B, R4, R5, and R6 are xe2x80x94CH3; wherein A is xe2x80x94CH2xe2x80x94Oxe2x80x94(CH2)2xe2x80x94NH2, and wherein R3 and R5 are xe2x80x94H, and wherein B, R4, and R6 are xe2x80x94CH3. The structure of these compounds is as follows. 
The present invention further contemplates compounds of the above structure with a primaryamino substituent on the pyrone ring, wherein A is at the 4-carbon atom and B is at the 3-carbon atom, preferably wherein A is selected from the group consisting of xe2x80x94CH2xe2x80x94NH2 and xe2x80x94CH2xe2x80x94Oxe2x80x94(CH2)2xe2x80x94NH2. More specifically, the invention contemplates compounds wherein A is xe2x80x94CH2xe2x80x94NH2, and wherein B, R3, R5, and R6 are xe2x80x94H, and wherein R4 is xe2x80x94CH3; wherein A is xe2x80x94CH2xe2x80x94NH2, and wherein B and R3 are xe2x80x94H, and wherein R4, R5, and R6 are xe2x80x94CH3; wherein A is xe2x80x94CH2xe2x80x94Oxe2x80x94(CH2)2xe2x80x94NH2, and wherein B and R3 are xe2x80x94H, and wherein R4, R5, and R6 are xe2x80x94CH3. The structure of these compounds is as follows. 
Additionally some of the new psoralens are primaryamino-benzene-linked psoralens comprising a primaryamino group linked to the benzene ring of the psoralen (5- and 8-carbon atoms) via an alkyl chain optionally containing oxygen and nitrogen atoms, and wherein the psoralen ring may have one or more alkyl, groups at other positions. The present invention further contemplates psoralen compounds with a primaryamino substituent on the benzene ring, comprising: a) a substituent A on the benzene ring, selected from the group consisting of: xe2x80x94(CH2)uxe2x80x94NH2, xe2x80x94(CH2)wxe2x80x94Jxe2x80x94(CH2)zxe2x80x94NH2, xe2x80x94(CH2)wxe2x80x94Jxe2x80x94(CH2)xxe2x80x94Kxe2x80x94(CH2)zxe2x80x94NH2, and xe2x80x94(CH2)wxe2x80x94Jxe2x80x94(CH2)xxe2x80x94Kxe2x80x94(CH2)yxe2x80x94Lxe2x80x94(CH2)zxe2x80x94NH2; wherein J, K, and L are independently selected from the group consisting of O and NH, in which u is 1 whole number from 1 to 10, w is a whole number from 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and b) substituents B, R1, R2, R4, and R5 on the benzene ring (the 5- or 8-carbon atom which does not have the primaryamino substituent), 3-, 4-, 4xe2x80x2-, and 5xe2x80x2-carbon atoms respectively, independently selected from the group consisting of xe2x80x94H and xe2x80x94(CH2)vCH3, where v is a whole number from 0 to 5, or a salt thereof; except c) when A is xe2x80x94CH2xe2x80x94NH2 and at the 8-carbon atom, one of B, R1, R4, and R5 must be xe2x80x94(CH2)vCH3. Where an element is xe2x80x9cindependently selectedxe2x80x9d, from a group, it means that the element need not be the same as other elements chosen from the same group. The structure of these compounds is as follows. 
The present invention further contemplates compounds of the above structure with a primaryamino substituent on the benzene ring, wherein A is at the 5-carbon atom and B is at the 8-carbon atom, preferably wherein A is selected from the group consisting of xe2x80x94CH2xe2x80x94NH2 and xe2x80x94CH2xe2x80x94Oxe2x80x94(CH2)2xe2x80x94NH2. More specifically, the invention contemplates compounds wherein A is xe2x80x94CH2xe2x80x94NH2, and wherein B, R1, R2, R4, and R5 are xe2x80x94H; wherein A is xe2x80x94CH2xe2x80x94Oxe2x80x94(CH2)2xe2x80x94NH2, and wherein B, R1, and R2 are xe2x80x94H, and wherein R4 and R5 are xe2x80x94CH3. The structure of these compounds is as follows. 
The present invention further contemplates compounds of the above structure with a primaryamino substituent on the benzene ring, wherein A is at the 8-carbon atom and B is at the 5-carbon atom, preferably wherein A is selected from the group consisting of xe2x80x94CH2xe2x80x94NH2 and CH2xe2x80x94Oxe2x80x94(CH2)2xe2x80x94NH2, and wherein when A is xe2x80x94CH2xe2x80x94NH2, at least one of B, R1, R4, and R5 are xe2x80x94(CH2)vCH3. More specifically, the invention contemplates compounds wherein A is xe2x80x94CH2xe2x80x94NH2, and wherein B, R1, and R4 are xe2x80x94H, and wherein R2 and R5 are xe2x80x94CH3; wherein A is xe2x80x94CH2xe2x80x94NH2, and wherein B and R1 are xe2x80x94H, and wherein R2, R4, and R5 are xe2x80x94CH3; wherein A is xe2x80x94CH2xe2x80x94Oxe2x80x94(CH2)2xe2x80x94NH2, and wherein B and R1 are xe2x80x94H, and wherein R2, R4, and R5 are xe2x80x94CH3. The structure of these compounds is as follows. 
The present invention also provides new routes to the synthesis of amninopsoralens and intermediates that may be useful for providing psoralens conjugated to a variety of other functional groups.
Without intending to be limited to any method of synthesis, the compounds of the present invention can be prepared by introduction of the amino functionality (or of a building block for said primaryamino group) in a protected form early on in the synthesis before the psoralen ring is fully constructed. This method provides new routes to the synthesis of primaryamino-pyrone-linked and benzene-linked psoralen s that allow more flexibility in the ring substituents than existing methods. This is exemplified in the synthesis of 3-aminomethyl-4xe2x80x2-methylpsoralen, 3-aminomethyl-4,4xe2x80x2,8-trimethylpsoralen, 3-aminomethyl-4,5xe2x80x2,8-trimethylpsoralen, 3-aminomethyl-4,4xe2x80x2,5xe2x80x2,8-tetramethylpsoralen, 3-(4-amino-2-oxa)butyl-4, 4xe2x80x2,8 trimethylpsoralen, 4-aminomethyl-4xe2x80x2-methylpsoralen, 4-aminomethyl(4xe2x80x2,5xe2x80x2,8-trimethylpsoralen, 4-(4-amino-2-oxa)butyl-4xe2x80x2,5xe2x80x2,8-trimethylpsoralen, 5-aminomethylpsoralen, 5-(4-amino-2-oxa)butyl-4xe2x80x2,5xe2x80x2dimethylpsoralen, 8-aminomethyl-4,4xe2x80x2,5-trimethylpsoralen aminomethyl4,5xe2x80x2-dimethylpsoralen), and 8-(4-amino-2-oxa)butyl-4,4xe2x80x2,5xe2x80x2-trimethylpsoralen (described in the examples below).
The present invention contemplates methods of inactivating pathogens in a biological composition, comprising, in the following order: a) providing, in any order, i) a compound selected from the group consisting of primaryamino-pyrone-linked psoralens and primaryamino-benzene-linked psoralens; ii) photoactivating means for photoactivating said compounds; and iii) a biological composition suspected of being contaminated with a pathogen which contains nucleic acid; b) adding said compound to said biological composition; and c) photoactivating said compound, so as to inactivate said pathogen. In one embodiment, the biological composition is a blood product. In a preferred embodiment, the blood product is either platelets or plasma. A preferred method of the present invention is performed in a blood bank or similar setting, wherein said compound is formulated in solution and said solution is contained in a blood compatible bag, and wherein said compound is added to said biological composition by flowing said biological composition through said bag. After treatment with the method, the blood product is suitable for its intended use.
A biological composition is defined as a composition originating from a biological organism of any type. Examples of biological compositions include, but are not limited to, blood, blood products (such as plasma, platelet preparations, red blood cells, packed red blood cells, and serum), cerebrospinal fluid, saliva, urine, feces, semen, sweat, milk, tissue, tissue samples, homogenized tissue samples, and any other substance having its origin in a biological organism. Biological compositions also include synthetic material incorporating a substance having its origin in a biological organism, such as a vaccine preparation comprised of alum and a pathogen (the pathogen being the substance having its, origin in a biological organism), cell culture medium, cell cultures, viral cultures, and other cultures derived from a biological organism.
A pathogen is defined as any agent which contains nucleic acid and is capable of causing disease in a human, other mammals, or vertebrates. Examples include microorganisms such as unicellular or multicellular microorganisms including but not limited to bacteria, viruses, protozoa, fungi, yeasts, molds, and mycoplasmas. The pathogen can comprise either DNA or RNA and this nucleic acid can be single stranded or double stranded.
The present invention contemplates that the photoactivating means comprises a photoactivation device capable of emitting a given intensity of a spectrum of electromagnetic radiation comprising wavelengths between 180 nm and 400 nm, preferably between 300 nm and 400 nm, and in particular, between 320 nm and 380 nm. It is preferred that the intensity is between 1 and 30 mW/cm2 and that the mixture is exposed to this intensity for between one second and thirty minutes [U.S. Pat. No. 5,593,823].
The present invention contemplates embodiments wherein said blood preparation is in a synthetic media. In one embodiment, the concentration of compound is between 0.1 xcexcM and 1000 xcexcM, preferably between 1 xcexcM and 500 xcexcM. In a preferred embodiment, the compound is added to said blood preparation at a concentration of between 10 xcexcM and 250 xcexcM.
The present invention contemplates embodiments of the methods where inactivation is performed without limiting (e.g. reducing) the concentration of molecular oxygen. Preferably, inactivation is performed without limiting the concentration of singlet oxygen that may be formed during the photoreaction step. Furthermore, there is no need for the use of cosolvents (e.g. dimethyl sulphoxide (DMSO)) to increase compound solubility.
In one embodiment, the present invention Contemplates methods of inactivating microorganisms in a blood product, wherein the compound is a primaryamino-pyrone-linked psoralen, comprising: a) a substituent A on the pyrone ring, selected from the group consisting of: xe2x80x94(CH2)uxe2x80x94NH2, xe2x80x94(CH2)wxe2x80x94Jxe2x80x94(CH2)zxe2x80x94NH2, xe2x80x94(CH2)wxe2x80x94Jxe2x80x94(CH2)xxe2x80x94Kxe2x80x94(CH2)zxe2x80x94NH2, and xe2x80x94(CH2)wxe2x80x94Jxe2x80x94(CH2)xxe2x80x94Kxe2x80x94(CH2)yxe2x80x94Lxe2x80x94(CH2)zxe2x80x94NH2; wherein J, K, and L are independently selected from the group consisting of O and NH, in which u is a whole number from 1 to 10, w is a whole number from 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and b)substituents B, R3, R4, R5, and R6 on the pyrone ring, 5-, 4xe2x80x2-, 5xe2x80x2- and 8-carbon atoms respectively, independently selected from the group consisting of xe2x80x94H and xe2x80x94(CH2)vCH3, where v is a whole number from 0 to 5; or a salt thereof.
Alternatively, the present invention contemplates embodiments of the method of inactivation, wherein the compound is a primaryamino-benzene-linked psoralen comprising: a) a substituent A on the benzene ring, selected from the group consisting of: xe2x80x94(CH2)uxe2x80x94NH2, xe2x80x94(CH2)wxe2x80x94Jxe2x80x94(CH2)zxe2x80x94NH2, xe2x80x94(CH2)wxe2x80x94Jxe2x80x94(CH2)xxe2x80x94Kxe2x80x94(CH2)zxe2x80x94NH2, and xe2x80x94(CH2)wxe2x80x94Jxe2x80x94(CH2)xxe2x80x94Kxe2x80x94(CH2)yxe2x80x94Lxe2x80x94(CH2)zxe2x80x94NH2; wherein J, K, and L are independently selected from the group consisting of O and NH, in which u is a whole number from 1 to 10, w is a whole number from 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and b) substituents B, R1, R2, R44 and R5 on the benzene ring, 3-, 4-, 4xe2x80x2-, and 5xe2x80x2-carbon atoms respectively, independently selected from the group consisting of xe2x80x94H and xe2x80x94(CH2)vCH3, where v is a whole number from 0 to 5; or a salt thereof, except c) when A is xe2x80x94CH2xe2x80x94NH2 and at the 8-carbon atom, one of B, R1, R4, and R5 must be xe2x80x94(CH2)vCH3.
In one embodiment of the method of inactivation, at least two of the compounds are present. The present invention contemplates embodiments where the compound is introduced either in aqueous solutions, such as water, saline, or a synthetic media, preferably a phosphate buffered media, non aqueous solutions such as alcohols, polyethylene glycols, or solvent mixtures with water, or in a dry formulation in which additives may be present. In one embodiment, the present invention contemplates a synthetic platelet storage media, comprising a glucose and magnesium free aqueous solution of: 45-120 mM sodium chloride; 5-15 mM sodium citrate; 20-40 mM sodium acetate; and 20-30 mM sodium phosphate. In a preferred embodiment, the aqueous solution comprises: approximately 86 mM sodium chloride; approximately 10 mM sodium citrate; approximately 30 mM sodium acetate; and approximately 26 mM sodium phosphate. The solution has a pH of approximately 300 milliosmolar/Kg. By not containing glucose or magnesium, the media is readily autoclaved.
The present invention contemplates embodiments wherein the compound may be introduced to the reaction vessel at the point of manufacture. Alternatively, the compound may be added to the reaction vessel at some point after the manufacture of, for example, a blood product. In one embodiment, a solution of the psoralen is provided in a biocompatible container that is attached to a disposable plastic set containing a unit of platelets or plasma. The psoralen solution is mixed with the blood product by passing said blood product through the container of psoralen and the resultant mixture is photoactivated with an illumination device suitable for uniform irradiation of blood bags [U.S. Pat. No. 5,593,823]. In a further embodiment, the residual psoralen and any low molecular weight psoralen photoproducts are removed from the solution [PCT publication WO 98/30327, hereby incorporated by reference].
In one embodiment, the blood product is admixed with the psoralen and the mixture is passed through a flow system where it is passed over a static light source resulting in photoactivation of said psoralen. The means of passing through said flow system includes but is not limited to gravity flow or metered flow, using a pump system.
The present invention provides new psoralens and methods of synthesis of new psoralens having enhanced ability to inactivate pathogens in the presence of ultraviolet light. The new psoralens are potentially effective against a wide variety of pathogens. The present invention also provides methods of using new and known compounds to inactivate pathogens in biological products to be used if vivo and in vitro, and in particular, blood products.
The inactivation methods of the present invention provide ex vivo methods of inactivating pathogens, and in particular, viruses, in blood products prior to use in vitro or in vivo. In contrast with previous approaches, the method requires only short irradiation times and there is no need to limit (e.g. reduce) the concentration of molecular oxygen or of singlet oxygen present or generated in the system.
In vivo use of a material is defined as introduction of the material or compound into a living human, mammal, or vertebrate. In vitro use of material or compound is defined as a use of the material or compound outside a living human, mammal, or vertebrate, where neither the material nor compound is intended for reintroduction into a living human, mammal, or vertebrate. An example of an in vitro use would be the analysis of a component of a blood sample using laboratory equipment. Ex vivo use of a compound is defined as using a compound for treatment of a biological material such as a blood product outside of a living human, mammal, or vertebrate, where that treated biological material is intended for use inside a living human, mammal, or vertebrate. For example, removal of blood from a human and introduction of a compound into that blood to inactivate pathogens is defined as an ex vivo use of that compound if the blood is intended for reintroduction into that human or another human. Reintroduction of the human blood into that human or another human would be in vivo use of the blood, as opposed to the ex vivo use of the compound.
The description of the invention is divided into the following sections: I) Compound Synthesis, II) Photoactivation Devices, III) Binding of Compounds to Nucleic Acid, IV) Inactivation of Nucleic Acid Containing Materials V) Preservation of Biochemical Properties of Material Treated, VI) Psoralen Conjugates and Other uses of Psoralens.
A. Psoralens as Photoactivation Compounds
The present invention contemplates those compounds described as psoralens: [7H -furo(3,2-g)-(1)-benzopyran-7-one, or b-lactone of 6-hydroxy-5-benzofuranacrylic acid], which are linear: 
and in which the two oxygen residues appended to the central aromatic moiety have a 1, 3 orientation, and further in which the furan ring moiety is linked to the 6-position of the two ring coumarin system. Psoralen derivatives are derived from substitution of the linear furocoumarin at the 3-, 4-, 5-, 8-, 4xe2x80x2-, or 5xe2x80x2-carbon atoms indicated in the above structure. For the purpose of this invention, substituents on the psoralen ring will be designated by the three ring structure consisting of the furan ring substituents (substituents linked to the 4xe2x80x2- and 5xe2x80x2-carbon atoms), the central benzene ring substituents (substituents linked to the 5- and 8-carbon atoms) and the pyrone ring substituents (substituents linked to the3- and 4-carbon atoms). More specifically, the present invention contemplates compounds with a primaryamino substituent on the 3- or 4-carbon atom herein referred to as primaryamino-pyrone-linked psoralens and compounds with a primaryamino substituent on the 5- or 8-carbon atom herein referred to as primaryamino -benzene-linked psoralens. In addition, the other of the 3- or 4-carbon atom of a primaryamino -pyrone-linked psoralen may contain an alkyl substituent. Similarly, the other of the 5- or 8-carbon atom of a primaryamino-benzene-linked psoralen may contain an alkyl substituent.
8-Methoxypsoralen (known in the literature under various names, e.g., xanthotoxin, methoxsalen, 8-MOP) is a naturally occurringlpsoralen with relatively low photoactivated binding to nucleic acids and low mutagenicity in an Ames assay. 4xe2x80x2-Aminomethyl-4,5xe2x80x2,8-trimethylpsoralen (AMT) is one of the most reactive nucleic acid binding psoralen derivatives, providing up to 1 AMT adduct per 3.5 DNA base pairs [S. T. Isaacs, G. Wiesehahn and L. M. Hallick, NCI Monograph 66: 21 (1984)]. However, AMT also exhibits significant levels of mutagenicity. A new group of psoralens was desired which would have the best characteristics of both 8-MOP and AMT: low mutagenicity and high nucleic acid binding affinity, to ensure safe and thorough inactivation of pathogens. One group of psoralens that has been synthesized and studied are primaryamino-furan-linked psoralens which are discussed in detail in U.S. Pat. No. 5,593,823. The compounds of the present invention are primaryamino-pyrone and primaryamino-benzene-linked analogs shown to be very effective at inactivation of R17, suggesting very high nucleic acid binding affinity.
Primaryamino-pyrone-linked psoralens are defined as psoralen compounds which have an xe2x80x94NH2 group linked to the 3-or 4-carbon atom of the psoralen by a hydrocarbon chain having a total length of 1 to 24 carbons, where 0 to 3 of those carbons are independently replaced by NH or O, and each point of replacement is separated from each other point of replacement by at least two carbons, and is separated from the psoralen by at least one carbon. Primaryamino-pyrone-linked psoralens may have additional substituents on the other of the 3- or 4-carbon atom and on the 5-, 8-, 4xe2x80x2-, and 5xe2x80x2-carbon atoms. Said substituents include but are not limited to xe2x80x94H and xe2x80x94(CH2)vCH3, where v is a whole number from 0 to 5. Compound I above gives the structure of primaryamino-pyrone-linked psoralens.
Primaryamino-benzene-linked psoralens are defined as psoralen compounds which have an xe2x80x94NH2 group linked to the 5- or 8-carbon at6m of the psoralen by a hydrocarbon chain having a total length of 1 to 24 carbons, where 0 to 3 of those carbons are independently replaced by NH or O, and each point of replacement is separated from each other point of replacement by at least two carbons, and is separated from the psoralen by at least one carbon. Primaryamino-benzene-linked psoralens may have additional substituents on the other of the 5- or 8-carbon atom and on the 3-, 4-, 4xe2x80x2-, and 5xe2x80x2-carbon atoms. Said substituents include but are not limited to xe2x80x94H and xe2x80x94(CH2)vCH3, where v is a whole number from 0 to 5. When the primaryamino substituent is on the 8-carbon atom, the present invention is limited in that at least one of the 3-, 5-, 4xe2x80x2- or 5xe2x80x2-substituents is xe2x80x94(CH2)vCH3. Compound IV above gives the structure of primaryamino-benzene-linked psoralens.
B. Synthesis of the Primaryamino-Pyrone-Linked Psoralens
Scheme 1 shows a method of synthesis of 3-halomethylcoumarins (1) and 4-halomethylcoumarins (4) useful for the preparation of the primaryamino-pyrone-linked compounds of the present invention. The compounds can be prepared from commercially available materials and converted to phthalimidomethyl-coumarins (2 and 5) and on to aminomethyl-pyrone-linked psoralens (3 and 6) by applying previously described methods [McLleod et al. Tetrahedron Lett. (1972) p. 237; Isaacs et al., Biochem. 16: 1058 (1977)] and further detailed in the examples. 
Longer chain aminoalkyl-pyrone linked psoralens can be prepared from the analogous haloalkylcoumarins (7 and 9 as shown in Scheme 2). While there are many ways of making the desired coumarins, many of them can most conveniently be prepared by the Pechman reaction [Organic Reactions, Vol VII, Chap 1, ed. Adams et al., Wiley, N.Y., (1953)] of resorcinols with the functionalized beta-keto esters. The desired beta-keto esters having a halide or halide synthon can be prepared by known methods [e.g., J. March, Advance Organic Chemistry, 3rd Ed., Wiley, (1985) pp437-440 and 824]. For example, Lambert et al., J. Org. Chem., 1985, 50, 5352; Gupta et al., J. Organomet. Chem. 1993, 444,1; Crombie et al., J. Chem. Soc Perk Trans I, 1987,333; Tremul Lozano, Span. Pat. 549788 A1 describe the preparation of desirable beta-keto-esters. The synthesis of such haloalkyl and hydroxyalkylcoumarins have been previously described [Zaniuk et al, Pol. Pat. PL 144435 B1, Fall et al, Heterocycles, (1995) 41, 647]. 
Alternatively, one can carry the protected alkylcoumarin (e.g. 7 where Y=halo, OH, OMe) through to a haloalkyl-pyrone linked psoralen and prepare the compounds of the present invention by applying some methods known in the art to functionalize haloalkylpsoralens. Examples of such functionalizations include the reaction of 4xe2x80x2-bromomethyl or chloromethylpsoralens (4xe2x80x2-BMT and 4xe2x80x2-CMT respectively) with ammonia, or potassium phthalimide (KPhth) followed by hydrazine to give AMT, as well as the reaction of the 4xe2x80x2-BMT and 4xe2x80x2-CMT with a variety of other amines. The identical reaction of 5xe2x80x2-bromomethyl or chloromethylpsoralen with KPhth is known [Kaufman U.S. Pat. No 4,294,822]. The reaction of 5-chloromethyl8-methoxypsoralen and 8-chloromethyl-5-methoxypsoralen with amines is known.
For the preparation of compounds of the present invention in which the linker between the primary amine and the psoralen contains one or more oxygen, one or more nitrogen, or both oxygen and nitrogen atoms, the preparation of such functionalized systems from haloalkylpsoralens has been thoroughly described previously for 4xe2x80x2- and 5xe2x80x2-primaryamino substituted psoralens [U.S. Pat. No. 5,654,443, incorporated by reference herein]. The same synthetic methods can be applied to the 3- and 4-primaryamino substituted psoralens as shown in the examples. Finally, the use of pseudo halides such as the methanesulfonyl group has been described such as in the reaction of 4xe2x80x2-(4-methanosulfonyloxy-2-oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen with sodium azide and subsequently converted into 4xe2x80x2-(4-amino-2-oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen. The same synthetic methods can be applied to the 3- and 4-primaryamino substituted psoralens as shown in the examples and typified in scheme 3 for the synthesis of 13 and 16, which have an oxygen atom in the primaryamino substituent chain. The synthesis starts from methoxyalkylcoumarins 7 and 9 above (where Y=OMe) which may be prepared from 7 and 9 (Y=halo, OH) or via direct coumarin synthesis. Conversion to psoralens 11a and 14a follows the same procedures described here in Scheme 1 and elsewhere. The psoralens are then de-methylated in a procedure that provides either the haloalkylpsoralen directly, or a hydroxyalkylpsoralen that is converted to a pseudohaloalkylpsoralen (11b and 14b below). By known procedures, set forth in the examples and elsewhere [U.S. Pat. No. 5,654,443] the haloalkylpsoralens are converted to the primaryamino-pyrone-linked substituted psoralens 13 and 16. 
C. Synthesis of Primaryamino-Benzene-Linked Psoralens
As described above for the primaryamino-pyrone-linked compounds, the desired primaryamino -benzene-linked compounds can be prepared by initial functionalization prior to formation of the psoralen ring system. Halomethyl coumarins (17 and 19 below) can be prepared by bromination of the appropriate coumarin as shown in Scheme 4 and then converted by procedures described in the examples and elsewhere into the desired aminomethylpsoralens (18 and 20 below) of the present invention. For primaryamino compounds linked to the 8 carbon atom, the 8-aminoloweralkylpsoralens and 8-aminoloweralkyl-4-loweralkylpsoralens are described in U.S. Pat. Nos. 4,328,239 and 4,269,851, hereby incorporated by reference. 
The above aminomethyl-benzene linked compounds and longer chain aminoalkyl-benzene linked psoralens (23 and 26 below) can be prepared by pre forming the analogous haloalkylcoumarins (22 and 25 as shown in Scheme 5). While there are many ways of making the desired coumarins, many of them can most conveniently be prepared by the Pechman reaction [Organic Reactions, Vol VII, Chap 1, ed. Adams et al., Wiley, N.Y., (1953)] of functionalized resorcinols with beta-keto esters. The desired resorcinols having a halide or halide synthon (21 and 24 below) can be prepared by known methods [see for example Makriyannis et al, U.S. Pat. No. 5,440,052; Seebach et al, Helv. Chim. Acta (1994) 77, 1673; Charalambous et al, J. Med. Chem (1992) 35, 3076; Elix et al, Aust. J. Chem. (1987) 40, 1841]. The haloalkylcoumarins can then be converted to protected aminoalkylcoumarins and taken on to the psoralens by methods described in the examples. 
Alternatively, one can carry the protected alkylcoumarin (e.g. 22 and 25 where Y=halo, OH, OMe) through to a haloalkylpyrone-linked psoralen and prepare the compounds of the present invention by methods known in the art to functonalize haloalkylpsoralens as shown in Scheme 6 for the synthesis of 28 and 30 below, which have an oxygen atom in the primaryamino substituent chain. The synthesis starts from methoxyalkylcoumarins 22 and 25 above (where Y=OMe) which may be prepared from 22 and 25 (Y=halo, OH) or via direct coumarin synthesis. Conversion to the psoralens, 27 and 29, follows the same procedures described here in Scheme 3 and elsewhere. By known procedures, set forth in the examples, the haloalkylpsoralens are converted to 28 and 30.
For the preparation of primary amino-benzene-linked psoralens in which the linker between the primary amine and the psoralen contains one, or more oxygen, one or more nitrogen, or both oxygen and nitrogen atoms, methods discussed above for the primaryamino-pyrone-linked psoralens can be used. 
D. Synthesis of Psoralen Conjugates.
The preparation of psoralen conjugates where the psoralen is linked to nucleotides, biotin, other intercalators, etc. has been described for the 4xe2x80x2-linked psoralens, for the 5-methyl linked-8-methoxypsoralen and for the 8-methyl linked 5-methoxypsoralen. While not being limited to any synthetic method to conjugate the psoralen onto another small molecule, protein, nucleic acid or material surface, typical methods for linking psoralens often entail one of three methods: 1) the bromomethylpsoralen is reacted with an amino or hydroxy function on the conjugated moiety; 2) the aminomethylpsoralen is reacted with an amide, urea or carbamate precursor (e.g., a succinamido group, or an isocyanate) on the conjugating moiety; and 3) the hydroxymethylpsoralen is reacted with an ester, or carbamate precursor on the conjugating moiety.
By the use of such known methods of conjugation, or alternative methods that may provide greater ease of preparation, one can prepare compositions comprising a psoralen linked through the pyrone ring to other nucleic acid binding moieties, to proteins, to nucleic acids, to fluorescent probes or other small molecules useful in diagnostics and to material surfaces.
Likewise, by the use of such known methods of conjugation, or alternative methods that may provide greater ease of preparation, one can prepare compositions comprising a psoralen linked through the benzene ring to other nucleic acid binding moieties, to proteins, to nucleic acids, to fluorescent probes or other small molecules useful in diagnostics and to material surfaces.
A variety of light devices may be useful for the photoactivation of compounds of the present invention and may be useful in the present methodology. Features of possible devices may be found in U.S. Pat. Nos. 5,593,823 and 5,683,661, hereby incorporated by reference. Additional features for possible uses of the compounds of this invention would include a means for passing a solution for inactivation through a light device such that the solution is sufficiently illuminated so as to inactivate pathogens within the solution. Said means may include gravity flow or metered flow, such as through a peristaltic pump or similar flow apparatus.
The present invention contemplates binding new and known compounds to nucleic acid, including (but not limited to) viral nucleic acid, bacterial nucleic acid, nucleic acid of lymphocytes, and nucleic acid of tissue cells such as smooth muscle cells. One approach of the present invention to binding photoactivation compounds to nucleic acid is photobinding. Photobinding is defined as the binding of photobinding compounds in the presence of photoactivating wavelengths of light. Photobinding compounds are compounds that bind to nucleic acid in the presence of photoactivating wavelengths of light. The present invention contemplates methods of photobinding with compounds of the present invention.
One embodiment of the method of the present invention for photobinding involves the steps: a) providing a photobinding compound of the present invention; and b) mixing the photobinding compound with nucleic acid in the presence of photoactivation wavelengths of electromagnetic radiation.
The invention further contemplates a method for modifying nucleic acid, comprising the steps: a) providing photobinding compound of the present invention and nucleic acid; and b) photobinding the photobinding compound to the nucleic acid, so that a compound:nucleic acid complex is formed.
The present invention contemplates treating a blood product with a photoactivation compound and irradiating to inactivate contaminating pathogen nucleic acid sequences before using the blood product. The present invention could also be applied to inactivation of other nucleic acid containing materials, such as lymphocytes, tissue cells, and solutions containing nucleic acids, for example solutions which have been amplified by polymerase chain reaction or a similar nucleic acid amplification technique.
A. Inactivation In General
The term xe2x80x9cinactivationxe2x80x9d is here defined as the altering of the nucleic acid in a material so as to render the nucleic acid incapable of replication. When the nucleic acid is that of a pathogen, the inactivation of the nucleic acid renders the pathogen incapable of replication. The inactivation of pathogens is detailed in U.S. Pat. No. 5,593,1823. In addition, inactivation may occur in any cell and the level of inactivation within a cell may be controlled by the level of photobinding of the psoralen to the nucleic acid. The level of photobinding can be controlled by varying either the dose of light used or the dose of the psoralen. The level of inactivation can be controlled, ranging from completely shutting down all cellular functions (high levels of photobinding) to shutting down proliferation of the cell while maintaining cellular functions (low levels of photobinding), i.e. the cell is still capable of transcribing the nucleic acid for the production of proteins. For example, a lymphocyte or tissue cell may be inactivated in that it can not replicate yet can still produce proteins and maintain biological function. This may also be referred to as inhibition of cellular proliferation rather than inactivation.
B. Inactivation of Potential Pathogens
In the case of inactivation methods for material to be used by humans, whether in vivo or in vitro, the detection method can theoretically be taken to be the measurement of the level of infection with a disease as a result of exposure to the material. The threshold below which the inactivation method is complete is then taken to be the level of inactivation which is sufficient to prevent disease from occurring due to contact with the material. It is recognized that in this practical scenario, it is not essential that the methods of the present invention result in xe2x80x9ctotal inactivationxe2x80x9d. That is to say, xe2x80x9csubstantial inactivationxe2x80x9d will be adequate as long as the viable portion is insufficient to cause disease. The inactivation method of the present invention renders nucleic acid in pathogens substantially inactivated. In one embodiment, the inactivation method renders pathogen nucleic acid in blood preparations substantially inactivated.
Without intending to be limited to any method by which the compounds of the present invention inactivate pathogens, it is believed that inactivation results from light induced binding of psoralens to pathogen nucleic acid. Further, while it is not intended that the inactivation method of the present invention be limited by the nature of the nucleic acid; it is contemplated that the inactivation method render all forms of nucleic acid (whether DNA, mRNA, etc.) substantially inactivated.
In the case of photoactivation compounds; modifying nucleic acid, it is preferred that interaction of the pathogen nucleic acid (whether DNA, mRNA, etc.) with the photoactivation compound causes the pathogen to be unable to replicate, such that, should a human be exposed to the treated pathogen, infection will not result.
xe2x80x9cSynthetic mediaxe2x80x9d is herein defined as an aqueous synthetic blood or blood product storage media. In one embodiment, the present invention contemplates inactivating blood products in synthetic media. This method may reduce product degradation during storage and permits the use of lower concentrations of photoactivation compounds.
The psoralen photoinactivation method inactivates nucleic acid based pathogens present in blood through a single procedure. Thus, it has the potential to eliminate bacteria, protozoa, and viruses as well. Had an effective decontamination method been available prior to the advent of the AIDS pandemic, no transfusion associated HIV transmission would have occurred. Psoralen-based decontamination has the potential to eliminate all infectious agents from the blood supply, regardless of the pathogen involved. Additionally, psoralen-based decontamination has the ability to sterilize blood products after collection and processing, which in the case of platelet concentrates could solve the problem of low level bacterial contamination and result in extended storage life. [J. Morrow et al., JAMA 266: 555-558 (1991); F. Bertolini et al., Transfusion 32: 152-156 (1992)].
A list of viruses which have been photochemically inactivated by one or more psoralen derivatives appears in Table 2 [From Table 1 of Hanson, C. V., Blood Cells 18:7 (1992)]. This list is not exhaustive, and is merely representative of the great variety of pathogens psoralens can inactivate. The present invention contemplates the inactivation of these and other viruses by the compounds described herein. The compounds of the present invention are particularly well suited for inactivating envelope viruses, such as the HIV virus.
C. Selecting Photoactivation Compounds for Inactivation of Pathogens
In order to evaluate a compound to decide if it would be useful in the methods of the present invention, two important properties should be considered: the compound""s ability to inactivate pathogens and the compounds effect on the suitability of the treated product for its intended use. A discussion of inactivation of pathogens other than the R17 model discussed below can be found in U.S. Pat. No. 5,593,823. This reference enables the selection criteria for use in pathogen inactivation of blood products for the compounds of the present invention. The screening technique used to evaluate the compounds of the present invention is to perform a bacteriophage screen; an assay which determines nucleic acid binding of test compounds. A screen of this type, an R17 screen, is described in detail in EXAMPLE 13, below.
The R17 bacteriophage screen is believed to be predictive of HIV inactivation efficiency, as well as the efficiency of compounds against many other viruses. It is a small, single stranded RNA phage. Without intending to be limited to any means by which the present invention operates, it is expected that shorter pieces of nucleic acid are harder to inactivate because they require a higher frequency of formation of psoralen adducts than do longer pieces of nucleic acid. Further, single stranded RNA pathogens are more difficult to inactivate because psoralens can neither intercalate between base pairs, as with double-stranded nucleic acids, nor form diadducts which function as interstrand crosslinks. Thus it is expected that when inactivation of R17 is achieved, these same conditions will cause the inactivation of many viruses and bacteria. More specifically, those compounds which exhibit  greater than 1 log inactivation of R17 (i.e.  greater than 90% kill) at a compound concentration in a test medium of 320 xcexcM or less are expected to be reasonable candidates for inactivation of pathogens in blood products.
Psoralens are useful in inactivation procedures because the reaction can be carried out at temperatures compatible with retaining biochemical properties of blood and blood products [Hanson, C. V., Blood Cells 18:7 (1992)]. The inactivation compounds and methods of the present invention are especially useful because they provide a means to inactivate pathogens while potentially retaining the suitability of the product for its intended use. The suitability of plasma may be measured by functionality of its protein components, either in whole plasma or after separation into plasma fractions. The suitability of platelets may be determined by methods and criteria similar to those used for establishing the suitability of storage and handling protocols.
Because of their affinity for nucleic acids and ability to covalently bind to nucleic acids, compounds of the present invention could be very useful when conjugated to other molecules. The conjugates may be formed by either chemical or photochemical attachment of the psoralen to another molecule, molecular fragment, or ligand. Materials to which psoralen may be conjugated include, but are not limited to, other nucleic acid binding moieties such as acridines or lexitropsins, proteins such as antibodies or receptor ligands, nucleic acids, small molecules useful in diagnostics such as fluorescent probes and biotin, and material surfaces.
The amino terminated chain of the psoralens of the present invention, or the halogen substituted intermediates indicated in EXAMPLES 6-9, are particularly suited to chemical attachment to other molecules. Such amino terminated compounds could be substituted for AMT in the following examples. In Wang Z. et al., J. Am. Chem. Soc. 117(20): 5438-5444 (1995), AMT is conjugated to a protected amino acid and subsequently used to prepare a psoralen:peptide conjugate. Such conjugates could be used to probe sequence specific proteinxe2x80x94nucleic acid interactions, or perhaps to selectively control gene expression. Similarly, psoralen may be conjugated to a nucleic acid oligonucleotide which is directed to a specific nucleic acid sequence (Vaghefi et al., PCT publication WO 92/02641). Such conjugates could be used as a control of gene expression or as a site specific probe of the nucleic acid sequence. The conjugation of the psoralen allows for a covalent photochemical attachment of the oligonucleotide to the target sequence. Other molecules conjugated to a psoralen starting from an amino terminal chain of the psoralen include, but are not limited to, biotin, fluorescent dyes, insulin, and lexitropsin, the uses of which are discussed in the references [U.S. Pat. Nos. 4,737,454, and 4,599,303, Biochem. and Biophys. Res. Comm. 141(2): 502-509 (1986), Anti-cancer Drug Design 9: 221-237 (1994)].
Any psoralen could potentially be conjugated to a nucleic acid photochemically. Such photochemical conjugates could be used as probes to specific nucleic acid targets. The photochemically conjugated psoralen could be prepared such that when the modified oligonucleotide pairs with the complementary target nucleic acid, the psoralen can crosslink the probe to the target strand with an additional UV light dose. Preparation and uses of such psoralen-oligonucleotide photochemical conjugates are described in U.S. Pat. Nos. 4,737,454, 4,599,303, and 5,532,146. Similarly, any material with which psoralen can interact and photobind could be photochemically conjugated to psoralen. For example, Bioconjugate Chem. 5(5): 463-467 (1994) give a method of photoreacting a psoralen compound with a polystyrene surface such as a microtiter plate. The psoralen can then be conjugated to another molecule such as an oligonucleotide, peptide, or biotin. While this reference uses psoralens containing a secondary amine, the primaryamino compounds of the present invention would also be useful using the conjugation schemes discussed in the references above.
Other possible uses of the compounds of the present invention include the inactivation of viruses for the purpose of preparing a vaccine, the inhibition of leukocytes to control proliferation yet maintain some function as a means of preventing graft vs. host disease in bone marrow transplants, and the inhibition of smooth muscle cells to control proliferation after injury, for example to prevent restenosis after balloon angioplasty. A discussion of the use of psoralens in vaccine preparation can be found in U.S. Pat. No. 5,106,619. A discussion of the use of 8-Methoxypsoralen for prevention of restenosis can be found in U.S. Pat. No. 5,354,774. These references are herein incorporated by reference.